What is ELISA?
ELISA Basics
ELISA standards for “enzyme-linked immunosorbent assay.” It has become a staple method for detecting soluble antigens in biomedical research. The ease with which ELISA can quantify multiple samples at once has made it a valuable tool for multiplex, high-throughput analysis of samples that have built in concentration standards.
The Principle of ELISA
Though simple, the concept of ELISA is quite elegant. Antigens of interest are trapped in a sandwich of antibodies. The bottom layer is anchored to a solid substrate so that the attached antigen is held in place. The top layer contains a reporter enzyme that performs a chemical reaction that produces a color compound. The more color appears in the solution, the more antigen is believed to be trapped in the sandwich. The color change in the solution can be quantified through spectrophotometry.
Since the solid substrate that anchors the bottom layer of antibodies can be a plastic petri dish, ELISA assays can be done on a 6-well or 96-well plate. The number of wells allows for multiple specimens and multiple experiments to be run at one time.
Common Antigens Detected by ELISA
ELISA is widely used to detect protein products, ranging from peptides to full-sized proteins, to glycoproteins. The limit of detection for what type of molecules can be measured by ELISA depends on how good the antibodies are at detecting that type of molecule. A limitation of ELISA is that it works well for only biomolecules that are soluble in an aqueous environment.
Direct vs. Indirect ELISA Assays
ELISA assays can be of two general forms, direct or indirect. Direct means that the antigen of interest is directly bound by the antibody that carries the enzyme reporter. Indirect means that the antibody that carries the enzyme reporter does not directly bind to the antigen of interest. Indirect methods use an extra antibody or molecule to bridge the antigen to the reporter antibody. The advantage of the indirect method is that it allows for more sites on the complex to contain an enzyme reporter. More reporters at each location mean a stronger signal is produced, otherwise known as amplifying the signal.
Importance of Blocking Buffers & Wash Buffers
Every binding assay in biomedical research has background noise that hinders the detection of a clear signal or a specific signal. The solid substrate on which the ELISA assay is built can bind nonspecifically to molecules in the solution. This can create a high background to noise ratio, which obscures the accuracy of the assay. Blocking buffers are used to coat the nonspecific binding regions on the solid substrate so that only the antigen attaches to the solid substrate because only binds specifically enough to not later was away. There is no one right blocking buffer since each antigen has different binding properties. A common blocking buffer is bovine serum albumin (BSA).
ELISA assays should be washed in between each step with a buffer that contains a light detergent. Common buffers are Tris-buffered saline (TBS) or Phosphate-buffered Saline (PBS). A common detergent is Tween-20, which is dissolved in the buffer at a concentration of 0.05%.